One of the more persistent objectives in molecular biology has been determining the nucleic acid sequence and relative abundance of individual species in heterogeneous mRNA populations. Methods for determining mRNA sequences typically involve analyzing the DNA sequence of single clones of a cDNA library, which are derived by enzymatic production of double-stranded cDNA from the mRNA. Methods for determining the relative abundance of mRNA species typically involve quantifying the hybridization of a defined nucleic acid sequence to a complementary sequence in the mRNA population. Analysis of samples containing a relatively low quantity of mRNA generally involves amplification prior to the application of methods for determining the sequence or relative abundance, of particular mRNA species. Amplification methods that proceed with linear kinetics during the course of the amplification reaction are less likely to introduce bias in the relative levels of different mRNAs than those that proceed with exponential kinetics (Shannon, U.S. Pat. No. 6,132,997).
In Van Gelder et al., U.S. Pat. No. 5,545,522, a process is described for amplifying a target nucleic acid sequence using a single primer-promoter, an oligonucleotide that has a sequence complementary to an RNA polymerase promoter linked to a sequence complementary to the target nucleic acid sequence. In an embodiment of this process, poly(A)+ mRNA is the target nucleic acid, with a primer-promoter having a 3′-terminal oligo(dT) sequence, for the amplification of “antisense RNA”, RNA transcripts that are complementary to the original mRNA. In this embodiment, cDNA is synthesized from the mRNA by extension of the annealed primer-promoter using reverse transcriptase; the RNA strand of the resulting mRNA:cDNA hybrid is partially hydrolyzed using RNase H; a second strand of DNA is synthesized from the cDNA by extension of the annealed mRNA fragments using DNA polymerase I (Gubler et al. (1983) Gene 25:263-269); and multiple copies of antisense RNA are synthesized from the second strand of DNA using an RNA polymerase. One problem with this method is that the 5′ ends of the mRNA, which become used as primers for second strand DNA synthesis, cannot be amplified and therefore cannot be identified. For 5′-terminal mRNA sequences to be included in an amplified product, an arbitrary sequence, a “sequence tag”, needs to be added to either the 5′ ends of the mRNA or the 3′ ends of the cDNA. This sequence tag provides a terminal priming site needed for amplification of the cDNA that was synthesized from the initial priming site, typically the 3′-terminal poly(A) of mRNA. Three general methods for providing a terminal priming site on mRNA or cDNA for the purposes of nucleic acid amplification are described below. Other methods based upon adding terminal polymer or oligomer tracts composed of the same nucleotide using enzymes such as terminal transfer or polyadenylate polymerase, “tailing methods”, are more applicable for cloning rather than amplifying nucleic acid molecules, and are thus not included.
In Kato et al., U.S. Pat. No. 5,597,713, a process is described for adding an arbitrary sequence to the 5′ ends of mRNA. In this process, mRNA is pretreated using a phosphatase to remove any terminal phosphates, the 5-′terminal cap is removed from the mRNA using a pyrophosphatase, and an oligonucleotide, having an arbitrary sequence composed of DNA and/or RNA, is added to the resulting 5′-terminal phosphate of the mRNA using T4 RNA ligase. In an embodiment of this process, cDNA having a 3′-terminal arbitrary sequence is synthesized from the ligated mRNA products by extension of an annealed oligo(dT) primer using reverse transcriptase. Since this process requires the performance of two hydrolytic steps on the mRNA, any contaminating hydrolytic activities in the enzymes and the alkaline reaction conditions can cause the loss of intact mRNA. In addition, T4 RNA ligase is less efficient with longer nucleic acid substrates.
In Dumas Milne Edwards et al. 1991 (Nucleic Acids Res. 19, 5227-5232) a process is described for amplifying 5′-terminal sequences of mRNA whereby an arbitrary sequence is added to the 3′ ends of cDNA. In this process, cDNA is synthesized from mRNA by extension of an annealed primer having a 3′-terminal oligo(dT) linked to a 41-nt arbitrary sequence using reverse transcriptase. After removing the mRNA from the resulting hybrid, an oligodeoxyribonucleotide, having a 44-nt arbitrary sequence, a 5′-terminal phosphate and a blocked 3′ end, is added to the 3′ ends of the cDNA using T4 RNA ligase. The ligated cDNA products, each with a different arbitrary sequence at each end, are amplified using PCR with primers derived from the 5′-terminal half of each arbitrary sequence. The resulting amplified products are purified and amplified using a second PCR this time with nested primers derived from the 3′-terminal half of each arbitrary sequence. For this process to work the optimum reaction conditions needed to be modified so that cDNA can be used as acceptor by T4 RNA ligase, resulting in the inefficient production of ligated cDNA as evidenced by the extensive exponential amplification that is required for their detection.
In Chenchik et al., U.S. Pat. No. 5,962,272, a process is described for the synthesis and cloning of cDNA corresponding to the 5′ ends of mRNA using a template-switching oligonucleotide that hybridizes to the 5′-terminal CAP of mRNA. The method comprises contacting RNA with a cDNA synthesis primer which can anneal to RNA, a suitable enzyme which possesses reverse transcriptase activity, and a template switching oligonucleotide under conditions sufficient to permit the template-dependent extension of the primer to generate an mRNA:cDNA hybrid. The template switching oligonucleotide hybridizes to the CAP site at the 5′ end of the RNA molecule and serves as a short, extended template for CAP-dependent extension of the 3′-end of the single stranded cDNA that is complementary to the template switching oligonucleotide. The resulting full-length single stranded cDNA includes the complete 5′-end of the RNA molecule as well as the sequence complementary to the template switching oligonucleotide, which can then serve as a universal priming site in subsequent amplification of the cDNA. The template switching oligonucleotide hybridizes to the CAP site at the 5′ end of the mRNA and forms basepair(s) with at least one nucleotide at the 3′ end of the cDNA of an mRNA-cDNA intermediate. Since this process is based upon the specific interaction with the CAP of an mRNA and the 3′ end of a cDNA in an mRNA-cDNA intermediate, it is unlikely to be applicable for adding terminal sequence tags to nucleic acid molecules that are single-stranded or are without a CAP structure.
The above is a cursory sampling of the methods that have been developed for the amplification of nucleic acid molecules. The person of skill in the art will be familiar with many of them and will also be familiar with their shortcomings. Some examples of the shortcomings include the sequence bias of exponential amplification and the inefficiency of single-stranded ligation; the narrow applicability to a few forms of RNA and DNA; and the requirement of a 5′-terminal CAP or an mRNA-cDNA intermediate. Notwithstanding the wide use of these amplification processes, a need exists for improvements. The research that is ongoing in this art is indicative of the search for a substantially universal method that can be broadly applied to unknown sequences in samples containing whole extractions of nucleic acids. Thus there is a need for a process that is capable of sensitive amplification of sequences from the entire mRNA, particularly from the 5′ ends.
The present invention seeks to meet these needs and other needs.